Embryonic development and the physiological

factors that coordinate hatching in domestic

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Embryonic development and the physiological factors that coordinate

hatching in domestic chickens

Veterinary Clinical Science, Royal Veterinary College, Hertfordshire, AL9 7TA, United
Kingdom
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Q. Tong
Division M3-BIORES: Measure, Model & Manage Bioresponses, Katholieke Universiteit
Leuven, Leuven, B-3001, Belgium
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C. E. Romanini
Division M3-BIORES: Measure, Model & Manage Bioresponses, Katholieke Universiteit
Leuven, Leuven, B-3001, Belgium
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V. Exadaktylos
Division M3-BIORES: Measure, Model & Manage Bioresponses, Katholieke Universiteit
Leuven, Leuven, B-3001, Belgium
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C. Bahr
Division M3-BIORES: Measure, Model & Manage Bioresponses, Katholieke Universiteit
Leuven, Leuven, B-3001, Belgium
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D. Berckmans
ANSES, Unit of Epidemiology and Welfare in Poultry and Rabbits, Ploufragan, 22440,
France
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H. Bergoug
ANSES, Unit of Epidemiology and Welfare in Poultry and Rabbits, Ploufragan, 22440,
France
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N. Eterradossi
Research and Development (R&D), Petersime NV, Zulte (Olsene), 9870, Belgium
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N. Roulston
Research and Development (R&D), Petersime NV, Zulte (Olsene), 9870, Belgium
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R. Verhelst
Veterinary Clinical Science, Royal Veterinary College, Hertfordshire, AL9 7TA, United
Kingdom
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I. M. McGonnell Show more
Veterinary Clinical Science, Royal Veterinary College, Hertfordshire, AL9 7TA, United
Kingdom
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T. Demmers

ABSTRACT

Embryonic growth and development is in uenced by both endogenous and exogenous

factors. The purpose of this review is to discuss the critical stages of chick embryonic
development in relation to functional maturation of numerous organ systems, the
acquisition of thermoregulation, and the hatching process. In addition, the mechanism of
hatching, including sound synchronization and hormonal and environmental stimulation,
will be discussed. Finally, the importance of effective hatching synchronization
mechanisms will also be highlighted.

INTRODUCTION

Hamburger and Hamilton (1992) described a series of stages encompassing the entire
period of chicken incubation (21 d) based on the external features. They de ned 3 main
phases: early, middle, and late stages. The formation of organs and systems occur
during the rst 2 stages, followed by the growth of organs and maturation of systems
during the late stage. Knowledge of the normal stages in chicken embryonic
development gives a deeper understanding of the developmental process and how
embryonic growth and the hatching process can be in uenced and manipulated by both
endogenous and exogenous factors. Although all vertebrate embryos are in uenced by
external environmental conditions, this is particularly true of embryos that develop
outside of the mother, as occurs in avian species. In avian systems, the environment is
tightly regulated by the parent(s) through the supply of nutrition to the egg and through
manipulation of incubation conditions in the wild (Reed and Clark, 2011). In nature, the
clutch hatches under the mother hen within a short hatch window (HW), which is de ned
as the time between the early-hatching and late-hatching chicks (Molenaar et al., 2010).
In contrast, during arti cial incubation, maternally derived components of incubation
control and sound communication are excluded. In the poultry industry, the HW can be as
long as 48 h, in part due to a difference in genetics and handling (i.e., storage conditions)
among the batches of eggs. As spread of hatch increases, the time of rst access to
feed and water also increases. A delay in access to feed for 1-d-old chicks impairs
posthatch growth (Careghi et al., 2005; van de Ven et al., 2009). Therefore, a short HW is
one way to improve animal welfare and achieve a more uniform ock.

The ability of the embryo to respond to the environment during incubation and adjust its
development is fundamental. The duration of incubation and time of hatching are mainly
regulated by embryonic physiology and behavior. Asynchronous hatching in clutches of
bird eggs is an environmentally dependent behavior and an adaptive phenomenon
among siblings (Rumpf and Tzschentke, 2010). Synchronization of hatching can occur
through coordinated vocalization and is also affected by physiological parameters such
as plasma thyroid and corticosterone (CORT) levels. The actual level of physiological
parameters that occur during the hatching process may predetermine the hatching time.
During the hatch, hormone systems act as critical endogenous effectors that transmit
environmental information to the cells, resulting in regulation of gene expression,
metabolism, and behavior (McNabb, 2007).

The purpose of this review is to highlight the timing of onset of embryo functional
systems: consciousness, mobility, thermoregulation, auditory, and vocalization based on
normal growth and development of the chick embryo throughout its incubation period
(Figure 1). We also discuss, in detail, some mechanisms and behaviors related to the
hatching process to understand effective ways to synchronize hatching. We provide
evidence to suggest that the optimization of incubation conditions by manipulating the
external environment can lead to a possible narrower HW.
 Timeline and onset of major functional milestones in embryonic development. HH =
Hamburger and Hamilton; REM: rapid-eye-movement; IP: internal pipping.
 Timeline and onset of major functional milestones in embryonic development. HH =
Hamburger and Hamilton; REM: rapid-eye-movement; IP: internal pipping.
STAGING EMBRYOS

In “A Series of Normal Stages in the Development of the Chick Embryo” (1951),

Hamburger and Hamilton developed and de ned speci c stages of morphogenetic
progression from the rst cleavage divisions through hatching. The classical Hamburger
and Hamilton (HH) Staging Table is widely used in classifying development of the chick
embryo into 45 stages (Hamburger and Hamilton, 1992). In their scheme, Hamburger and
Hamilton used different prominent morphological changes and developmental features
to classify different phases of development. The initial stages (1 to 6) are characterized
by the development of the primitive streak. The middle stages (7 to 14) are de ned
primarily by the number of somites and related features. The late developmental stages
(15 to 45) are identi ed by several typical morphological features and grouped by a
series of standard measurable features. The morphological changes of limbs and
visceral arches are used to determine stages 18 to 40. However, toward the end of
development, the chick does not undergo further morphological changes and only
increases in size; therefore, these late stages require identi cation by objective
measurements (such as the length of beak and third toe).

Eyal-Giladi and Kochav (1976) provided the rst stage-by-stage procedure to describe the
morphogenetic development of the early chicken embryo during preoviposition. In the
Eyal-Giladi and Kochav 14-stage procedure (Eyal-Giladi and Kochav, 1976), a stage I
embryo is a blastodisc with less than 5 or so cleavage divisions and a stage XIV embryo
has a completed hypoblast that is generally equivalent to stage 1 in the HH Staging
Table. Thus, combining these 2 staging schemes gives a complete and comprehensive
staging reference throughout chick development.

Although we recognize that the temporal rate of development differs among different
species, strains, and even individuals, the external features of any embryo within any
given stage will be almost identical (Sellier et al., 2006). With the detailed descriptions of
a normal table as a reference, it is possible to identify the actual stage at which prenatal
activation of functional systems occurs and the proper time of embryonic response. This
knowledge can be applied not only to scienti c research but also in the poultry industry
for evaluating the effect of preincubation handling on embryo development; estimation of
embryonic mortality at different stages when performing fresh egg and candling
breakouts; de ning normal and abnormal embryo development; and evaluating the
impact of other factors (hen age, strain, and shell quality) on fertility and hatchability.
These reference tables ensure that accurate, consistent, and repeatable data are
obtained among laboratories and represent a standard for industry quality assessment.

DEVELOPMENT OF EMBRYO FUNCTIONAL SYSTEM

The development of organs start early in chick embryonic development, and all
functional organs are well developed before hatch. Initial development of the chick
embryo is rapid: by h 44 of incubation, the heart is beating and vascular systems are
linked. By the end of the third day of incubation, limb buds for the wings and legs are
visible and the auditory pit is established. Formation of reproductive organs,
differentiation of sex, and beginning of voluntary movements take place on d 5 and 6. By
d 7, the heart is completely enclosed in the thoracic cavity and feather buds are forming.
By d 10, the beak hardens and toes are fully formed (stage 36 of HH when the length of
beak and third toe are started to use as an indicator for embryonic staging). On
embryonic d 14, the embryo is moving into position for hatching and turns its head
toward the large end of egg. Rapid-eye-movement (REM) is detected at d 15, which is
related to high levels of forebrain activation in the chick embryo and signals the time the
embryo enters a growth phase before hatching (Mellor and Diesch, 2007; Mellor, 2010).
Furthermore, it is shown that chicken maternal calls at this stage can stimulate the
embryonic brain to a waking-like state in advance of hatch (Balaban et al., 2012).

Development of Mobility and Nervous Systems

Development of the central nervous system (CNS) in the chick embryo begins on the
second day of incubation and matures before hatching on d 21. Embryonic movement
represents the development and interaction of skeletal, muscular, and nervous systems,
and these commence on d 3 of development. Embryonic limb movement serves an
essential role in joint cavity formation and cartilage and bone formation during skeletal
development. Restricted early embryonic movement can result in positional deformity, a
reduced heart rate, reduced blood ow, oxygen and nutrient delivery, and therefore
decrease growth (Lamb et al., 2003; Pitsillides, 2006). The onset sequence of activity in
neurological structures underlying sensory mechanisms is: tactile by d 6, proprioceptive-
vestibular by d 8 to10, taste possibly around d 12, auditory by d 12 to14, vision by d 18,
and olfactory by d 20 (Rogers, 1995). With the peripheral, visceral, spinal, and brain nerve
tracts developing, growing, and maturing, motor behaviors begin as spontaneous
movements at d 3 (controlled individual limb, neck, and head movements) to the highly
coordinated movements that prepare the body to the right position for hatching (Wu et
al., 2001).

Heat Production

Energy changes and balance within an avian egg are represented by embryonic heat
production (HP) which affects egg shell temperature. Body temperature is primarily
controlled by the rate of HP and exchange with the environment. Mechanisms of HP, heat
conservation, and heat loss are controlled by the central nervous system.
Thermoregulation as an open loop system and a closed feedback control system occurs
at d 15 and 20, respectively, when the embryo can react to the changes of incubation
temperature (Tzschentke, 2008).

Different developmental stages show different HP rates during incubation (Table 1). The
developmental stage-related HP of the chicken embryo gradually increases from d 9 to
16 and then reaches a plateau phase from d 17 to 19 (Janke et al., 2004). Embryonic
growth rate declines shortly before hatching so that the metabolic rate usually plateaus
before hatching in precocial birds (domestic chicken; Vleck and Vleck, 1987). At the end
of the plateau phase the embryo penetrates the chorioallantoic and inner shell
membranes and begins pulmonary respiration to supply the growing embryo with
su cient oxygen. The HP increases continuously from internal pipping until hatch (Wu et
al., 2001; Tzschentke, 2008). Qualitatively similar developmental patterns of HP were
found in all precocial and altricial bird species investigated (Tzschentke and Rumpf,
2011). Embryonic body temperature measured in the allantoic uid also follows the
same developmental pattern of HP (Janke et al., 2002). There are 2 possible
explanations for this phenomenon. One is that HP is calculated by O2 consumption and
CO2 production under constant incubation temperature; therefore, increasing metabolic
rate and oxygen consumption may cause an increase in body temperature. The second is
the possibility that increasing body temperature is due to an increased thermoregulatory
set-point and this in turn leads to an increased HP. However, at the late stage of
incubation, lung respiration (a mechanism of heat loss) is considered to be more
effective in controlling body temperature than HP mechanisms via adjusting the
respiration rate and tidal volume (Tzschentke, 2008). Different strains also show different
HP rates (Table 1). The HP has a tendency to be higher in layers than in broilers, possibly
because protein synthesis (which requires energy) is faster in layers than in broilers
(Romijn and Lokhorst, 1960; Muramatsu et al., 1987; Muramatsu et al., 1990; Sato et al.,
2006). Differences in HP have also been noticed between different strains of broilers
(Tona et al., 2004; De Smit et al., 2005).

Mean values of heat production and body temperature during incubation in Ross 308, Ross 508, and White
Leghorn chickens (Janke et al., 2004)

Heat production (W∙kg−1)  Body temperature (°C) 

Incubation
Ross Ross White   Ross Ross White
day 
308  508  Leghorn  308  508  Leghorn 
11  0.33  0.46  0.44    37.6  37.8  37.9 
12  0.90a  0.87a  0.57b    38.2a  38.2a  37.9b 
13  1.24a  1.35a  0.82b    38.6a  38.6a  38.2b 
 Heat production (W∙kg−1)  Body temperature (°C) 
Incubation
Ross Ross White   Ross Ross White
day 
308  508  Leghorn  308  508  Leghorn 
14  1.54a  1.81a  1.17b    38.9a  39.0a  38.3b 
15  2.09a  2.23a  1.47b    39.4a  39.3a  38.7b 
16  2.15  2.57  1.89    39.4ab  39.6a  39.0b 
17  2.52  2.67  2.22    39.6  39.6  39.3 
18  2.61  2.49  2.17    39.7a  39.6a  39.3b 
19  2.74a  2.68a  2.12b    39.7a  39.6a  39.2b 
20  4.20a  3.99a  2.18b    40.5a  40.5a  39.0b 
Heat production (W∙kg−1)  Body temperature (°C) 
Incubation
Ross Ross White   Ross Ross White
day 
308  508  Leghorn  308  508  Leghorn 
11  0.33  0.46  0.44    37.6  37.8  37.9 
12  0.90a  0.87a  0.57b    38.2a  38.2a  37.9b 
13  1.24a  1.35a  0.82b    38.6a  38.6a  38.2b 
14  1.54a  1.81a  1.17b    38.9a  39.0a  38.3b 
15  2.09a  2.23a  1.47b    39.4a  39.3a  38.7b 
16  2.15  2.57  1.89    39.4ab  39.6a  39.0b 
17  2.52  2.67  2.22    39.6  39.6  39.3 
18  2.61  2.49  2.17    39.7a  39.6a  39.3b 
19  2.74a  2.68a  2.12b    39.7a  39.6a  39.2b 
20  4.20a  3.99a  2.18b    40.5a  40.5a  39.0b 
Heat production (W∙kg−1)  Body temperature (°C) 
Incubation
Ross Ross White   Ross Ross White
day 
308  508  Leghorn  308  508  Leghorn 
11  0.33  0.46  0.44    37.6  37.8  37.9 
12  0.90a  0.87a  0.57b    38.2a  38.2a  37.9b 
13  1.24a  1.35a  0.82b    38.6a  38.6a  38.2b 
14  1.54a  1.81a  1.17b    38.9a  39.0a  38.3b 
15  2.09a  2.23a  1.47b    39.4a  39.3a  38.7b 
16  2.15  2.57  1.89    39.4ab  39.6a  39.0b 
17  2.52  2.67  2.22    39.6  39.6  39.3 
18  2.61  2.49  2.17    39.7a  39.6a  39.3b 
19  2.74a  2.68a  2.12b    39.7a  39.6a  39.2b 
 Heat production (W∙kg−1)  Body temperature (°C) 
Incubation
Ross Ross White   Ross Ross White
day 
308  508  Leghorn  308  508  Leghorn 
20  4.20a  3.99a  2.18b    40.5a  40.5a  39.0b 
Heat production (W∙kg−1)  Body temperature (°C) 
Incubation
Ross Ross White   Ross Ross White
day 
308  508  Leghorn  308  508  Leghorn 
11  0.33  0.46  0.44    37.6  37.8  37.9 
12  0.90a  0.87a  0.57b    38.2a  38.2a  37.9b 
13  1.24a  1.35a  0.82b    38.6a  38.6a  38.2b 
14  1.54a  1.81a  1.17b    38.9a  39.0a  38.3b 
15  2.09a  2.23a  1.47b    39.4a  39.3a  38.7b 
16  2.15  2.57  1.89    39.4ab  39.6a  39.0b 
17  2.52  2.67  2.22    39.6  39.6  39.3 
18  2.61  2.49  2.17    39.7a  39.6a  39.3b 
19  2.74a  2.68a  2.12b    39.7a  39.6a  39.2b 
20  4.20a  3.99a  2.18b    40.5a  40.5a  39.0b 

Auditory Development

Several studies report that the auditory development in birds is precocious during
incubation (Konishi, 1973; Friauf and Lohmann, 1999; Rubel and Fritzsch, 2002).
However, it is not clear when the embryo is able to detect and encode natural ambient
sound, and what are the ranges of characteristic frequencies (CF) and threshold levels
for sensitivity. The cytological differentiation of hair cells in the domestic chick occurs
between d 8 and 10, and by d 10 the middle ear apparatus is also well-formed
(Whitehead and Morest, 1985a,b). Synaptic contact in the hair cells of the chicken basilar
papilla appear on d 10 (Rebillard and Pujol, 1983; Whitehead and Morest, 1985a,b), and
central synapses between primary afferents and second-order auditory neurons in
nucleus magnocellularis are functional around d 12 (Jackson et al., 1982; Pettigrew et al.,
1988). The response of cochlear ganglion neurons to sound are tested, and results
indicate that in domestic chickens (Gallus gallus L. f. domestica) the ontogeny of hearing
begins with endogenous rhythms around the period of peripheral synaptogenesis
between d 13 and 14 (HH stages 39 to 40). Cochlear primary afferent neurons cannot
encode frequency information from the natural sound environment until the sensitivity
occurs and matures at stage 42. From d 15 to 16 (stages 41 to 42), cells responded to
external sound at a relatively high threshold (86.0 ± 10.9 dB) and wide range of
frequencies (50 to 5,000 Hz). The CF threshold levels for airborne sound stimulation
decrease in older embryos (stages 43 to 44) to 60.5 ± 18.2 dB sound pressure level
(which is a logarithmic measure of the effective sound pressure of a sound relative to a
reference value) and CF became restricted to frequencies ≤ 2,300 Hz. Airborne CF
threshold levels decreasing with embryonic stage indicate that sound transfer improves
during embryonic development (Jones et al., 2006).

Previous studies show morphological and biochemical alterations in brainstem auditory

nuclei and hippocampus of domestic chicks given early prenatal sound stimulation by
species-speci c sound at 65 dB from d 10 until hatch (Chaudhury et al., 2006, 2009;
Chaudhury and Wadhwa, 2009). Increased dendritic arborization and probably enhanced
synaptogenesis could be indirectly attributed to consolidation and maintenance of
synapses consequent to adherent activity induced by prenatal sound stimulation (Alladi
et al., 2002). These effects indicate that prenatal auditory stimulation played an
important role in chick learning and memory (Kauser et al., 2011). It is possible that
prenatal auditory stimulation may also play a role in the synchronization of hatching
(Veterany et al., 1999b; Vergne and Mathevon, 2008).

MECHANISM OF HATCHING

Hatching Behavior

Hatching behavior is well described in literature and begins on d 16 or 17 of incubation in

the chick embryo (Oppenheim, 1970, 1972; Provine, 1971, 1972; Oppenheim et al., 1978;
Pittman et al., 1978). The embryo turns its body to the correct position: a folded neck and
head under the air space membrane on d 18. Such neck folding is maintained after both
internal and external pipping and until the chick escapes from the shell once hatching is
complete. From d 19 and 20, hatching behavior is more active and vigorous head
movements cause its beak to penetrate the membrane (internal pipping). The allantois,
which previously served as its mode of gas exchange, begins to dry up as the chick
starts pulmonary respiration. A period of relative inactivity follows, punctuated by
occasional vigorous whole-body and head movements about 24 h before hatching. Then,
the chick continues to push its head outward using the sharp horny structure on the
upper beak (egg tooth) and the hatching muscle on the back of the neck to open a spot
on the eggshell (external pipping). This is followed by another quiescent period until the
hatching behavior on d 20 or 21. A series of back thrusts with the beak and egg tooth
against the shell enlarges the hole made by external pipping, which, together with
repeated partial body rotations achieved by stepping movements of the feet against the
shell, lead to the top of the shell being loosened su ciently to be pushed off. However, it
is not clear what causes the sudden appearance of the required active behaviors after
the period of inactivity that follows external pipping. External in uences such as vocal-
auditory interactions between the hen and chick, between chicks in the same clutch, or
both, may in uence the timing. Another assumption is that hatching initiates once the
partial pressure of CO2 (pCO2) threshold in the air cell is reached and increases
metabolic demand. Blood pCO2 and air cell pCO2 are increasing with embryonic
development. From d 14 to 18, air cell pCO2 increases from 15 to 34 mmHg (Bruggeman
et al., 2007). Furthermore, high ambient CO2 can cause higher blood and air cell pCO2 in
the early stage of incubation (De Smit et al., 2006; Bruggeman et al., 2007). Up to d 19,
oxygen supply occurs only via blood circulating through the chorioallantoic membrane
(Mortola, 2009). From d 19 to 20, however, the chick pierces the air-space membrane of
the egg with its beak (internal pipping), which allows pulmonary respiration to begin.
Thereafter, until hatching is complete at about d 20 or 21, oxygen supply occurs via both
the chorioallantoic membrane and the lungs. The oxygen contributed via the
chorioallantois declines to low levels during this period, but the pulmonary contribution
compensates with a 1.5- to 2-fold rise. This is aided at approximately d 19 or 20 by the
chick penetrating the eggshell with its beak (external pipping) and thereby gaining
access to atmospheric air. There needs to be an overall balance between delivery of
oxygen by these means and an increase in oxygen demand by the chick as it grows, as
well as the marked increase in demand associated with prehatching and hatching
behavior (Menna and Mortola, 2002; Mellor and Diesch, 2007).

Vocalization and Hatching Synchronization

Hearing sensitivity, sound production, and transmission between eggs and maternal
birds are necessary for the acoustic communication at late stage of embryonic
development. Bird’s embryos are able to produce and perceive sounds before hatching,
which plays a vital role in synchronize hatching. The avian embryo produces the rst calls
before internal pipping, and the real vocalization gradually develops a species-speci c
sound after penetration of the inner egg membrane (Rumpf and Tzschentke, 2010). Then
the embryos begin to regularly produce clicking sounds due to the egg tooth tapping
against the eggshell. Clicks are accompanied by the development of breathing and
respiration movements that are not a real vocalization but sounds controlled by the
syrinx (Vince and Salter, 1967; Terskova, 1975; Hoyt and Rahn, 1980; Veterany et al.,
1999b). The acoustic communication by clicking sounds is an essential factor for
hatching synchronization in many bird species by either accelerating or decelerating the
hatching time of embryo (Vince, 1966a,b, 1973, 1984; Brua, 2002).

Clicking sounds are well studied in quails and Muscovy ducks, but not in the domestic
chicken. It has been reported that some sounds play a special role for acoustic
communication in ducks. Responses to acoustic stimuli are increasing the rate of bill
clicking, foot movements, eye opening, vocalization, or a combination of these. The best
hearing frequency for clicking sound communication in the last quarter of incubation is a
range of 300 to 600 Hz (Gottlieb, 1965; Heaton, 1972; Gottlieb, 1982; Gottlieb, 1983;
Hochel et al., 2002). In the domestic chicken, hatching occurred earlier in chicks exposed
to clicking sounds than in control chicks treated with no sound stimulation or maternal
calls. These results suggest that chicks accelerate hatching in response to internally
generated clicks but not to external stimuli such as a maternal call (White, 1984).

Based on prenatal clicking and breathing rates, metabolic rate may also be another
mechanism by which hatching is synchronized in Muscovy duck embryos. When the
second embryo within a clutch begins to click within a certain species-speci c range,
embryos adapt their clicking rates to those of others, which results in a common rhythm.
Toward hatching, embryos click more and more regularly with an increased synchronized
clicking rate (Vince, 1973; Rumpf and Tzschentke, 2010). However, the actual
mechanism underlying clicking sound communication is not yet clear. It is also not clear
whether an embryo can just hear its neighbor(s) or all the embryos in the clutch. One
hypothesis is that most of the sounds the embryo hears within the prenatal period are
bone-conducted and not air-conducted. Because the head of the embryo has contact
with the eggshell at that time, bone conduction to the embryo’s ear is possible. Besides
clicking sounds, several other acoustic factors have been investigated in the hatching
process: other calls from embryos, calls and bill clicking from the maternal hen, and
music were all tested in laboratory experiments. However, these studies suggest that
none of the acoustic signals mentioned improved hatching synchronization or served as
a hatching releasing signal (Vince, 1969; Veterany et al., 1999a; Boonstra et al., 2010;
Rumpf and Tzschentke, 2010; Reed and Clark, 2011). However, the communication
between embryo and maternal parent by any other means (vocalizations, movements,
thermal and chemical signals) is still very important for the time of hatching and
epigenetic adaption (Toien et al., 1986; Field et al., 2007; Grans and Altimiras, 2007).

Thyroid and Adrenal Hormone Changes

The thyroid and adrenal glands are functional by the end of the rst week of incubation in
the chicken embryo. Thyroid hormone and glucocorticoid secretion are stimulated by the
thyroid-stimulating hormone (TSH) and adrenocorticotropic hormone (ACTH), which
have been reported to appear at d 4.5 and 7, respectively (De Groef et al., 2008). The
peripheral glands are controlled and regulated when the hypothalamo-pituitary axis is
formed around mid-incubation. Toward the end of incubation, endocrine axes undergo
profound changes: not only stimulating general growth and differentiation of the chick
embryo but also preparing it for its life outside the egg by regulating processes such as
yolk sac retraction, the onset of lung respiration, hatching, and the initiation of
endothermic responses. Thyroid hormones (TH) and glucocorticoid levels (CORT) are the
most important hormones that act as stimulants for the hatching process. There are 2
types of TH responses in avian species embryos: developmental and metabolic.
Thyroxine (T4) is a prohormone that is converted into bioactive 3,3′,5-triiodothyronine
(T3), which circulates into cells and tissues. Plasma T3 is detectable at 9.5 d of
incubation. Data from RIA measurement of T3 and T4 concentrations in chick embryo
plasma show that they increase on d 15 before peaking. After internal pipping, T4 levels
reduce, whereas T3 reaches a maximum during hatch (Figure 2). After hatch, both T4 and
T3 return to low levels (Reyns et al., 2003; Lu et al., 2007). This suggests that the thyroid
hormones play an important role in the hatching process between the start of pulmonary
respiration and hatching. In addition, higher TH concentrations have been linked to a
shorter HW. In turkey embryos, TH are being elevated to affect maturation and survival
functions during the nal stages of incubation, and consequently affecting hatching time
(Christensen et al., 2002).
 The 3,3′,5-triiodothyronine (T3), thyroxine (T4), and corticosterone (CORT)
concentration (ng/mL) in plasma from 11-d-old embryos to newly hatched chicks (Scott
et al., 1981; Reyns et al., 2003; Sechman et al., 2006). IP = internal pipping; H = hatch.
 The 3,3′,5-triiodothyronine (T3), thyroxine (T4), and corticosterone (CORT)
concentration (ng/mL) in plasma from 11-d-old embryos to newly hatched chicks (Scott
et al., 1981; Reyns et al., 2003; Sechman et al., 2006). IP = internal pipping; H = hatch.
Plasma CORT is also related to embryonic development and a higher concentration leads
to the initiation of chick’s hatching process (Decuypere et al., 1991). The CORT can
stimulate growth hormone (GH) secretion on embryonic d 12 in the chicken, and the
secretion of CORT during embryonic development is modulated by T3 and T4 (Mashaly,
1991; Bossis and Porter, 2000). Plasma CORT concentration can be detected by d 10;
then pituitary control of the adrenal function becomes important at about d 14. From d
10 to 14, the levels of CORT uctuate slightly and then increase sharply by d 16. From
this time, plasma CORT remains relatively stable through d 18 with an increasing trend
until d 20 (Figure 2; Kalliecharan and Hall, 1974, 1976; Scott et al., 1981).

Prenatal stress has the potential to cause serious consequences to production

agriculture and animal well-being. Therefore, absence of stress or the ability to cope with
stress is a potential indicator for positive animal welfare (Archer et al., 2009).
Glucocorticoids (cortisol and CORT) are stress hormones and re ect the activity of
adrenal glands (Mostl and Palme, 2002).

Environmental factors can in uence circulating hormone levels, thus affecting metabolic
functions and the hatching process (Kuhn et al., 1984). However, the exact physiological
mechanism by which this occurs is not clear. The CO2 concentration is known to be a
gaseous environmental stimulus, which could alter plasma hormones. Hypoxic
conditions stimulate CORT secretion at late stages of incubation (Blacker et al., 2004).
Compared with ventilated incubation (CO2: 0.1%), chicken embryos in nonventilated
incubation (CO2 reached 1% or 1.5% at d 10) had higher plasma CORT and T3 levels at
pipping, and the HW was advanced by 10 to 15 h as well as narrowed. It was
demonstrated that increased CO2 during early stages of incubation enhanced embryonic
growth, chick quality, and broiler performance (De Smit et al., 2006; Bruggeman et al.,
2007; Tona et al., 2007; De Smit et al., 2008). In addition, high CO2 concentration in the air
cell during the hatching phase can trigger the embryo into pipping and stimulate embryos
to emerge from the egg, thereby reducing the HW (Molenaar et al., 2010). However, both
the mechanism and impact on chick quality and posthatch performance is not yet clear.

CONCLUSIONS AND PERSPECTIVES

A synchronized birth is important for any species that produces many offspring in a
clutch or litter as it creates an ideal environment for survival. Altering the environment of
a developing organism might change the developmental trajectories of some
physiological regulation systems and may alter the time of hatching and may also
subsequently affect the characteristic maturity or quality of the young during the rst few
days’ life (Tzschentke and Plagemann, 2006). For chicks, it is believed that simply
reducing HW and forcing chicks to hatch without being well-developed may have a
negative effect on chick quality and subsequent performance. Therefore, to achieve
better chick quality and welfare, optimal incubation conditions have to be applied and the
embryo response has to be considered. Besides incubation conditions (temperature,
humidity, gas pressure) and embryonic physiological parameters, other environmental
factors are also important to determine the actual hatching times, such as order of
ovulation, thermal microenvironments of eggs in incubation period, and intraclutch
communication.

It is possible to manipulate environmental conditions to change circulating hormone

levels that lead to an optimal hatchability and HW. To create an incubation system that
will automatically lead to synchronized hatching, some key factors should be considered:
1) preincubation conditions; 2) incubation conditions; 3) maturity and sensitivity of
embryo functional system; 4) the period and features of external stimuli; and 5) species-
speci c ability to synchronize. Combination of incubation conditions and
microenvironment of eggs might be a way to achieve ideal ock uniformity.

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